Immunotherapeutic strategies incorporating intralesional (IL) therapy to elicit tumor specific immune responses can serve as a non-surgical option for cutaneous neoplasms. These strategies haven been shown to induce both local and systemic tumor regressions. Intratumoral injection of dendritic cells (DCs), IL-2, and GM-CSF and treatment with adjuvant Bacille Calmette-Guérin (BCG) or toll-like receptor (TLR) agonists have been shown to enhance systemic anti-tumor immunity in both melanoma tumor-bearing mice and in patients with advanced melanoma [Triozzi et al., Cancer 89:2646-2654 (2000); Pilon-Thomas et al., J Immunother 29:381-387 (2006); Guo et al., Int J Cancer 120:2418-2425 (2007); Kaufman et al., Ann Surg Oncol 17:718-730 (2010); Kidner et al., J Immunother 35:716-720 (2012)]. Dendritic cells are the most potent antigen presenting cells (APCs) and can prime an immune response by T cells that have not been exposed to the antigen previously [Small et al., J Clin Oncol 18:3894-3903 (2000)].
In the 20th century, several uses for fluorescein analogs emerged. The compounds have been used as textile dyes, biological stains, building blocks for non-volatile memory devices, thermoimaging substrates and food and cosmetics coloring. For example, erythrosine (FD&C No. 3) and partially iodinated erythrosine (D&C Nos. 11 and 12) are used as food, drug and cosmetic dyes. A particular tetraiodo xanthene, rose bengal, has been used for visualization of ocular disease and, in radiolabeled form, as a medical diagnostic for liver function, appearing in the United States Pharmacopeia in 1965.
Use of rose bengal (RB) as a biological stain by ophthalmologists and in liver function studies became common in the 20th century [Norn, Acta Ophthalmol 48:546-559 (1970); Delprat Arch. Int. Med. 32:401-410 (1923)]. RB can kill both microorganisms and cancer cells as a photodynamic sensitizer or even without laser activation for metastatic melanoma and ovarian cancer [Banks et al., J Appl Bact 58:391-400 (1985); Koevary, Int J Physiol Pathophysiol Pharmacol 4:99-107 (2012); Thompson et al., Melanoma Res 18:405-411 (2008); Wachter et al., Proc. SPIE 4620:143-147 (2002)].
RB can pass through the cell membrane and accumulate in the lysosomes of tumor cells and autolyse tumor cells within 30-60 minutes, while it is excluded from normal cells [Wachter et al., Proc. SPIE 4620:143-147 (2002)]. Notably, IL therapy of PV-10 (10% rose bengal in PBS; Provectus Biopharmaceuticals, Inc., Knoxville, Tenn.) has been shown to elicit tumor-specific immunity in human studies [Thompson et al., Melanoma Res 18:405-411 (2008); and Thompson et al., Ann Surg Oncology 22:2135-2142 (2015)] manifested in un-injected bystander lesion regression. Further studies revealed that IL PV-10 treatment can induce T-cell mediated tumor-specific immune responses in MT901 breast cancer and in B16 melanoma mouse models [Toomey et al., PloS one 8:e68561 (2013)]. However, the underlying mechanisms remain unknown.
Thus, rose bengal has been disclosed as an ablative agent patented for tumor destruction (U.S. Pat. No. 8,557,298). A novel action of post rose bengal ablation is the ability of immune system components to recognize tumor tissue in situ in the treated mammal. These immune system components have been found to induce a systemic immune system response in the ablation-treated mammal.
A number of strategies have been proposed to induce immune responses as a treatment strategy in cancer. These approaches generally consist of removing diseased tissue from the host and manipulating that tissue to target, treat or expand in size or number, useful immune system components prior to readministration of the immune components to the host.
For example, vaccines comprised of dendritic cells pulsed ex vivo with tumor antigens and expansion of natural killer cells are under investigation (U.S. Pat. No. 8,597,946). Antigen- and non-antigen-based antibodies have been manufactured and manipulated ex vivo to target tumor tissue (U.S. Pat. No. 8,153,120, US 2004/0161413 A1). Notably, a combination of non-myeloablative chemotherapy and adoptive transfer of expanded T-cells has been reported to result in sustained clinical responses in late-stage cancer patients [Pilon-Thomas, J. Immunother 35(8):615-620 (2012)]. These strategies are independent of endogenous immune tissue and are manufactured ex vivo from extracted tumor tissue or peripheral blood for administration to patients. Additionally, the use of non-myeloablative chemotherapy adds additional time and cost to the treatment, and can enhance the possibility of increased treatment-based morbidity.
Dying cancer cells can release soluble molecules known as damage-associated molecular pattern molecules (DAMPs), which are mainly recognized by pattern recognition receptors (PRRs) [Zitvogel et al., Cell 140:798-804 (2010)]. Particular DAMPs can serve as powerful immunological adjuvants for cancer therapy [Kroemer et al., Ann Rev Immunol 31:51-72 (2013); Krysko et al., Nature Rev Cancer 12:860-875 (2012)]. These DAMPs include several members of the heat shock protein (HSP) family, the 5100 proteins, ATP, IL-1α and high mobility group box 1 (HMGB1), also known as amphoterin [reviewed by Panzarini et al., PloS one 9:e105778 (2013)].
HMGB1 is an abundant protein bound to DNA in almost all eukaryotic cells. Its putative receptors include the receptor for advanced glycation end-products (RAGE), Toll-like Receptor-2 (TLR2), TLR4 and T cell immunoglobulin-3, (TIM-3) (Taguchi et al., Nature 405:354-360 (2000); Park et al., J Biol Chem 279:7370-7377 (2004); Chiba et al., Nature Immunol 13:832-842 (2012)]. HMGB1 has a membrane-bound form and also can be secreted into the extracellular space as a cytokine-like factor. It is secreted from activated immune cells such as macrophages and dendritic cells (DCs) after its acetylation, or can be released by necrotic, apoptotic and autophagic cancer cells as a DAMP [Scaffidi et al., Nature 418:191-195 (2002); Bonaldi et al., EMBO J 22:5551-5560 (2003); Kazama et al., Immunity 29:21-32 (2008); Thorburn et al., Cell Death Differ 16:175-183. (2009)].
HMGB1 plays an important role in the activation of endothelial cells, promotion of angiogenesis, immune cell migration, and initiation of inflammation [Lotze et al., Nature Rev Immunol 5:331-342 (2005)]. Although HMGB1 has been shown to contribute to tumor metastasis and neoangiogenesis, its release by dying tumor cells can lead to the activation of DCs to prevent tumor progression [Apetoh et al., Nature Med 13:1050-1059 (2007); Curtin et al., PLoS Med 6:e10 (2009)].
There are many ways known to isolate, bank, expand, target, and retreat cancer patients with tissues designed to stimulate immune system anti-tumor activity. Some strategies such as PROVENGE® (SIPULEUCEL-T) and adoptive transfer start with patient peripheral blood, lymphoid or tumor tissue. However, none of these strategies uses an intralesional ablation of tumor tissue to enhance the quality of endogenous immune components in situ prior to their removal from the patient. The post removal treatment strategies can be patient-specific for personalized therapies.
Immunoglobulins (antibodies) and sometimes vaccines to common diseases have been used to enhance an immune response for tumor treatment in a wider patient population. Such antibodies and vaccines are more generalizable to patients from whom they are not necessarily isolated. For example, antibody design using endogenous ligands is generally applicable to a wide variety of patients and these ligands are often discovered after probing a system with a stimulus to elucidate an antibody that is more generalizable.
Similarly, antibodies can be engineered to mimic naturally occurring events in endogenous immune responses to cancer. For example, the anti-CTLA-4 (cytotoxic T lymphocyte-associated antigen 4) monoclonal antibodies ipilimumab and tremelimumab are designed to counter down-regulation of the immune system by blocking CTLA-4 activity and thus augment T-cell response against cancer. Similarly, monoclonal antibodies such as pidilizumab, nivolumab, lambrolizumab and pembrolizumab bind to PD-1 (programmed death 1) receptor to counter down-regulation of the immune system and augment T-cell responses to cancerous tumors. Initial work with antibodies to the PD-1 ligands, PD-L1 and PD-L2, such as BMS-936559, MEDI4736 and atezolizumab (MPDL3280A) to PD-L1, also indicate inhibition of down-regulation of the immune system and an augmented T-cell response against cancer.
Alternative approaches utilize substances that stimulate certain components of the immune system (i.e., up-regulation or down-regulation), including administering non-specific cytokines (such as interleukin-1, -2, or -12; “IL-1”, IL-2”, or “IL-12”; interferon-alpha or gamma, “IFN-α” and “IFN-γ”; granulocyte macrophage colony stimulating factor, “GM-CSF”), or that attempt to provoke a tumor-specific immune response.
As disclosed hereinafter, it is believed that removal of tissue containing immune cells from a host or tumor cells treated with a halogenated xanthene, such as rose bengal in PV-10, can be used to make general antibodies or personalized therapies. These components could be isolated after exposure to the ablative compound.
Intralesional administration of tumor tissue with a halogenated xanthene (such as that of PV-10) releases tumor antigens that stimulate tumor-specific immune cells found in peripheral blood, local lymphoid tissue or tumor tissue after ablation but not in significant levels in these tissues of a placebo-treated subject. The local antigen-presenting cells are thereby pre-loaded with tumor debris and can be valuable in the treatment of disease on their own merit.
The disclosure that follows shows that IL PV-10 injection can elicit tumor-specific immune responses in illustrative patients with melanoma and in melanoma-bearing mice. It is further found that an underlying mechanism is that IL injection of PV-10 into melanoma tumors leads to the release of HMGB1 and activation of immune cells for the induction of tumor-specific immunity. The induced tumor responses in vivo and in vitro cascade in tumor and immune system tissues whose activated cells can be banked and reintroduced, or expanded and then reintroduced using techniques known in the art to treat or inhibit further episodes of the cancer.
Additionally these locally used ablative agents can be used in mammalian or in vitro studies as a tool to identify an antibody specific to the ablated cells, or as a tool for identification prior to cloning of useful biologic material for the treatment of the cancer. These antibodies can be useful as a more general treatment of cancer either alone or after cloning and manufacturing according to techniques known in the art. The immune components so generated and isolated from peripheral blood, spleen, tumor, or lymph nodes are capable of responding to tumor both in mice and in people and can be detected within about 1 day or more following intralesional injection.